As a method of quantitating a microorganism, there have been conventionally mainly used a method involving culturing a microorganism in a preliminarily estimated selection medium and measuring the number of microbial cells and a method involving culturing a microorganism in a liquid selection medium and measuring the optical density or absorbance. The following methods have also been used for the identification operation for a microorganism needed in detecting the microorganism in a specimen, for example, a method involving identification thereof through morphologic observation, Gram staining, and microbiological characteristics such as oxygen requirement, sugar-assimilating properties and growing condition in a medium; a method involving determination thereof by a DNA-DNA homology test; and a detection method using a monoclonal antibody to a microbial surface antigen. However, these methods require time and skill and therefore have presented a problem from a standpoint of rapidity and simplicity.
In recent years, gene amplification methods including a PCR method have been used in a wide range of fields as techniques for detecting traces of nucleic acids. These methods have advantages capable of leading to speed-up and simplification, including no mandatory requirement for culturing a microorganism contained in a specimen and capability of directly handling a specimen as a sample. Thus, the methods have been subjected to the investigation of application to the quantitation and detection of a microorganism.
As an example where the PCR method has been applied to the analysis of a microorganism, a method is known for quantitating a bacterium by a PCR method which uses the total DNA as a target sequence and universal primers (Patent Document 1). Methods using 16S rDNA as a target have also been achieved. Known examples thereof include a method for quantitative analysis by a PCR method using 16S rDNA as a target sequence (Patent Document 2), a method for analysis of an intestinal bacteria by a PCR method using 16S rDNA as a target sequence (Patent Document 3), and a method for detection of a bacterial strain of the genus Lactobacillus, a bacterium causing turbidity of beer (Patent Document 4). However, these methods have had a problem that they cannot be used as alternatives to a conventional method which has been conventionally employed because detection sensitivity is not achieved to the extent obtained with the culture method. By way of example, performing the method for quantitative analysis as disclosed in Patent Document 2 requires a large amount of template DNA corresponding to a microbial count of 105/μl or more, which makes the method impractical. The low detection sensitivity is probably due to the low number of copies (amount of template), of the total DNA or 16S rDNA providing as a template for the PCR in the microorganism. Since DNA is known to remain even after the dying-out of a microorganism, these methods only quantitate and detect dead and live microorganisms together, which also has posed a problem that they are difficult to precisely quantitate and detect a microorganism in a live state (Non-patent Document 1).
As examples of application of a PCR method to the analysis of a microorganism, attempts have also been made to perform methods using mRNA as a target sequence; known examples thereof include quantitative analysis of a lactic acid bacterium in feces, employing mRNA as a target sequence (Non-patent Document 2). Methods for detecting cancer cells are also known which use as target sequences mRNAs specific to cancer cells in specimens (Patent Documents 5 and 6). However, even these methods have not provided detection sensitivity to the extent that they can replace the conventional method as quantitation methods. Specifically, the detection limit of the quantitative analysis as shown in Patent Document 2 is only 103.5 or more cells/g of feces; the analysis method has not been able to be used as an alternative to the conventional culture method in view of detection sensitivity. In addition, these methods target mRNAs of genes unique to the microorganisms, and have been unsuitable for detection in a specimen to be tested containing a large variety of microorganisms because of problems such as complicated primer design and reduced specificity.
Accordingly, the development of a method has been awaited which provides detection sensitivity to the same extent as conventional detection methods while being a rapid method using a PCR method or the like and which further can precisely quantitate and detect a microorganism in a live state.
To improve sensitivity, it is possible to change the design of a target so that the target can be present more stably or in more abundance in cells. However, such a stable target is probably unfavorable for the purpose of detecting only a live microorganism, considering that it is suspected to remain long also in a dead cell thereof. Thus, it is not easy to simultaneously achieve the detection of only live cells and sufficiently high detection sensitivity.
Also, it has been known that rRNA accounts for about 85% of the content of RNA in a cell and has a multicopy number and that rRNA is stable compared to mRNA because it forms a complex with protein. rRNA is also reported to be detected for on the order of 48 hours after microbial death (Non-patent Document 3) and therefore has been commonly believed to be unsuitable for detection of a microorganism in a live state (Non-patent Document 1).
Patent Document 1: Japanese Patent Laid-Open No. 2002-238585
Patent Document 2: Japanese Patent Laid-Open No. 2003-259879
Patent Document 3: Japanese Patent Laid-Open No. 2001-112485
Patent Document 4: Japanese Patent Laid-Open No. 10-210980
Patent Document 5: Japanese Patent Laid-Open No. 10-248600
Patent Document 6: International Publication WO 00/17395 pamphlet
Non-patent Document 1: J Food Prot, vol. 67, No. 4: (2004)
Non-patent Document 2: FEMS Microbiology Letters, vol. 231: 125-130 (2004)
Non-patent Document 3: Appl. Environ. Microbiol., vol. 64, No. 11: 4264-4268 (1998)
An object of the present invention is to provide a method of quantitatively analyzing a microorganism, which can achieve detection sensitivity to the extent of being capable of replacing a conventional culture method and more precise detection of the microorganism in a live state.